Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis

• Published in: Phytochemistry (Oxford) 2007-03, Vol.68 (6), p.785-796
• Main Authors: Zhou, Xue-Rong, Robert, Stanley S., Petrie, James R., Frampton, Dion M.F., Mansour, Maged P., Blackburn, Susan I., Nichols, Peter D., Green, Allan G., Singh, Surinder P.
• Format: Article
• Language: English
• Subjects: Algal Proteins - genetics; Algal Proteins - metabolism; Base Sequence; Biological and medical sciences; Blotting, Southern; Chemical constitution; Chromatography, Gas; Cloning, Molecular; complementary DNA; conserved sequences; DNA, Complementary - chemistry; DNA, Complementary - genetics; docosahexaenoic acid; Docosahexaenoic Acids - metabolism; eicosapentaenoic acid; eicosatetraenoic acid; Eukaryota - enzymology; Eukaryota - genetics; fatty acid composition; Fatty Acid Desaturases - genetics; Fatty Acid Desaturases - metabolism; Fatty Acids, Unsaturated - metabolism; Fundamental and applied biological sciences. Psychology; Gas Chromatography-Mass Spectrometry; gene expression regulation; Gene Expression Regulation, Enzymologic; Gene isolation; Isoenzymes - genetics; Isoenzymes - metabolism; long chain fatty acids; Long chain polyunsaturated fatty acids; Metabolism; Metabolism. Physicochemical requirements; Microalgae; Models, Biological; molecular cloning; Molecular Sequence Data; nucleotide sequences; Pavlova; Pavlova salina; Phylogeny; Plant physiology and development; Polymerase Chain Reaction; polyunsaturated fatty acids; Reverse Transcriptase Polymerase Chain Reaction; sequence analysis; Sequence Analysis, DNA; stearoyl-CoA desaturase; Δ4-desaturase; Δ5-desaturase; Δ8-desaturase
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2006.12.016

The fatty acid Δ4, Δ5 and Δ8-desaturase genes were cloned from marine microalga Pavlova salina, and the encoded enzymes produced omega-3 long chain polyunsaturated fatty acids (LC-PUFA) eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in yeast cells and/or transgenic seeds, respectively, with appropriate substrates. The marine microalga Pavlova salina produces lipids containing approximately 50% omega-3 long chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Three cDNA sequences, designated PsD4Des, PsD5Des, PsD8Des, were isolated from P. salina and shown to encode three front-end desaturases with Δ4, Δ5 and Δ8 specificity, respectively. Southern analysis indicated that the P. salina genome contained single copies of all three front-end fatty acid desaturase genes. When grown at three different temperatures, analysis of fatty acid profiles indicated P. salina desaturation conversions occurred with greater than 95% efficiency. Real-Time PCR revealed that expression of PsD8Des was higher than for the other two genes under normal growth conditions, while PsD5Des had the lowest expression level. The deduced amino acid sequences from all three genes contained three conserved histidine boxes and a cytochrome b 5 domain. Sequence alignment showed that the three genes were homologous to corresponding desaturases from other microalgae and fungi. The predicted activities of these three front-end desaturases leading to the synthesis of LC-PUFA were also confirmed in yeast and in higher plants.